U.S. patent number 6,043,229 [Application Number 08/982,747] was granted by the patent office on 2000-03-28 for highly fermentable resistant starch.
This patent grant is currently assigned to Cerestar Holding B.V.. Invention is credited to Francis Bornet, Jozef Victor Jean-Marie Coppin, Bernd Wolfgang Kettlitz, Harald Wilhelm Walter Roper.
United States Patent |
6,043,229 |
Kettlitz , et al. |
March 28, 2000 |
Highly fermentable resistant starch
Abstract
The present invention discloses that retrograded starch having
more than 55% resistant starch with >50% chains of DP 10-35
gives rise to a significantly higher amount of n-butyrate
production under conditions simulating the human colon. It is
expected that such an increased n-butyrate production will diminish
the development of colon diseases notably of colon cancer.
Inventors: |
Kettlitz; Bernd Wolfgang
(Bonheiden, BE), Coppin; Jozef Victor Jean-Marie
(Denderleeuw, BE), Roper; Harald Wilhelm Walter
(Brussels, BE), Bornet; Francis (Rhode Saint-Genese,
BE) |
Assignee: |
Cerestar Holding B.V. (La Sas
Van Gent, NL)
|
Family
ID: |
10803869 |
Appl.
No.: |
08/982,747 |
Filed: |
December 2, 1997 |
Foreign Application Priority Data
Current U.S.
Class: |
514/60; 536/102;
536/123; 536/103; 536/105 |
Current CPC
Class: |
A61K
31/718 (20130101); A61P 1/12 (20180101); A23C
9/1544 (20130101); A23L 29/35 (20160801); C08B
30/12 (20130101); C12P 19/16 (20130101) |
Current International
Class: |
A23C
9/152 (20060101); A23C 9/154 (20060101); A23L
1/09 (20060101); A61K 31/716 (20060101); A61K
31/718 (20060101); C08B 30/00 (20060101); C08B
30/12 (20060101); C12P 19/16 (20060101); C12P
19/00 (20060101); C12P 019/16 (); C08B
030/20 () |
Field of
Search: |
;514/60
;536/102,103,105,123 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
5470391 |
November 1995 |
Mallee et al. |
|
Foreign Patent Documents
|
|
|
|
|
|
|
564 893 |
|
Oct 1993 |
|
EP |
|
688 872 |
|
Dec 1995 |
|
EP |
|
91/07106 |
|
May 1991 |
|
WO |
|
Other References
Carbohydrate Polymers, vol. 21, No. 2/3, 1993, Barking GB, pp.
195-203, XP000387697 F. Bornet: "Technological treatments of
cereals." .
"Novelose" resistant starch; National Starch and Chemical Co.;
Circle 34, p. 220; 1994. .
"Functional Fiber Debut Marks the Birth of a Whole New Class of
Ingredients"; Food Engineering: p. 27; Oct. 1994. .
"Resistant Starch"; Mike Croghan; NR. 3; pp. 33-35; 1995..
|
Primary Examiner: Wilson; James O.
Assistant Examiner: Owens; Howard
Attorney, Agent or Firm: Pillsbury Madison & Sutro
LLP
Claims
We claim:
1. A partially degraded, debranched and retrograde starch
comprising more than 55% (w/w) pancreatine resistant starch
(RS),
wherein the resistant starch consists essentially of alpha-glucans
having a chain length distribution of more than 50% in the range
between (DP) 10 and 35 and a differential scanning chromatography
(DSC) melting temperature below 115.degree. C.
2. A starch according to claim 1 wherein the resistant starch has a
DSC peak melting temperature between 90 and 114.degree. C.
3. A partially degraded, debranched and retrograde starch according
to claim 1,
wherein said partially degraded retrograde starch produces higher
amounts of short chain fatty acids than retrograded products
derived from amylose starches having less than 50% chain length
distribution in the range between DP 10 and 35 when fermented by
microorganisms normally found in the human hind gut.
4. A starch according to claim 1 wherein it is retrograded
debranched maltodextrin with a dextrose equivalent below 10.
5. A starch according to claim 4 wherein the maltodextrin is
derived from potato starch or tapioca.
6. A starch according to claim 4 wherein the maltodextrin is
derived from a starch selected from the group consisting of a high
amylose starch, maize, wheat, tapioca and pea starch.
7. A starch according to claim 1, 2 or 3 consisting of a debranched
retrograded acid thinned starch.
8. A starch according to claim 7 obtained from a starch selected
from the group consisting of potato, tapioca, maize, pea or wheat
starch.
9. A method for obtaining a starch according to claim 1
characterised by the following steps:
a) thinning of starch,
b) enzymatic debranching of the thinned starch,
c) inactivation of the enzyme,
d) drying of the composition.
10. A method according to claim 9 containing the following
steps:
a) a maltodextrin with a DE below 10 is dissolved in water,
b) the pH is adjusted and the solution is cooled to a optimum
temperature for the activity of debranching enzymes,
c) enzyme is added and the mixture is incubated,
d) the enzyme is inactivated,
e) the mixture is spray-dried
f) the resistant starch is collected and optionally grinded.
11. A food preparation characterised in that it contains up to 10%
of a starch-based composition according to claim 1.
12. A starch according to claim 3, wherein said short chain fatty
acid comprises n-butyrate.
13. A starch according to claim 1, wherein said pancreatine
resistant starch is in an amount of at least 60% (w/w).
Description
TECHNICAL FIELD
The present invention relates to a starch composition containing a
high proportion of so-called "resistant starch" (RS). The
composition of the resistant starch is further characterised by a
specific chain length distribution of the RS-fraction and by a
relatively low specific Differential Scanning Chromatography (DSC)
melting peak temperature. The composition furthermore shows a
specific fermentation pattern resulting in an increased level of
n-butyrate.
BACKGROUND OF THE INVENTION
It has been known for some years that a part of the starch
contained in the human diet can pass the small intestine without
being digested. This fraction of the food starch is called
resistant starch. Different forms of starch have been found to be
resistant to digestion. A classification of resistant starches has
been given by Englyst and Cummings (Am. J. Clin. Nutr. (1987) 45
423-431). These authors distinguish between three types of
resistant starches:
Type 1. Physically indigestible starch e.g. partially milled grains
and seeds,
Type 2. Resistant starch granules e.g. raw potato, green
banana,
Type 3. Retrograded starch e.g. cooled-cooked potato, bread, and
cornflakes.
Effective enrichment of food with RS is possible by addition of
processed starch containing a large percentage of retrograded
structures. Starch is composed of amylose and amylopectin. The
extent of retrogradation is known to be a function of the amylose
content. Heating and cooling of amylose gives rise to resistant
starch. Due to the branched structure of amylopectin the amount of
resistant starch which is formed is decreasing with an increase in
the amount of amylopectin in starch. The amount of RS can however
be increased by debranching the amylopectin prior to heating. In
view of the above high amylose (maize) starches have been chosen as
the primary source of resistant starch for the first commercial
high RS-products.
Carbohydrates which are not enzymatically digested in the small
intestine reach the colon where they are fermented by the anaerobic
microflora. Such carbohydrates include non-starch polysaccharides,
resistant starch (RS), indigestible oligosaccharides and endogenous
polysaccharides from mucus. The undigested starch fraction reaches
the colon where it becomes a substrate for microbial fermentation.
Besides gas production (H.sub.2, CH.sub.4, CO) different short
chain fatty acids (SCFA) are formed depending on the type of
carbohydrate.
The major end products of bacterial carbohydrate breakdown are
short-chain fatty acids (SCFA: acetate, propionate and n-butyrate).
SCFA are rapidly taken up by the colonic epithelial cells.
Propionate and acetate are released by the basolateral membrane to
the portal circulation and may have an effect far from their
production site. n-Butyrate serves as energy yielding substrate in
the colonocytes and additionally affects several cellular functions
e.g. proliferation, membrane synthesis and sodium absorption.
Acetate, propionate and n-butyrate are the main SCFA produced from
indigestible oligo- and polysaccharides the relative amounts of
these fatty acids depend on the type of carbohydrate. SCFA are
produced in the proximal colon in an average ratio of
acetate:propionate: n-butyrate equivalent to 60:25:10 and in
amounts of mmol/L. This ratio however is riot constant but is
determined by the kind of substrate fermented. It has been shown
both in vitro and in vivo that the fermentation of starch yields
high levels of n-butyrate. The observations that ceacal SCFA levels
are decreased by raw potato starch (Mallett et al. (1988) Brit. J.
Nutr. 60, 597-604; Levrat et al.(1991), J. Nutr. Biochem.2, 31-36;
Mathers et al., (1991) Brit. J. Nutr.66, 313-329) but increased by
high amylose corn starch underline that different forms of RS have
different effects in terms of n-butyrate production in the
colon.
According to Wyatt and Horn (1988) J. Sci. Food Agric. 44, 281-288,
RS-fractions of retrograded pea and corn starch respectively show
quantitative differences in in vitro fermentation but without
qualitative changes in SCFA composition. Six different raw starches
also showed different in vitro fermentation kinetics. At the same
time the molar n-butyrate proportion was not altered. Several
independent in vivo animal studies confirm this. Thus the source of
RS is important for the fermentability and hence for the amount of
n-butyrate obtained but apparently not for the relative amount.
Compared with indigestible polysaccharides such as arabinogalactan,
xylan and pectin, RS produces a significantly larger molar amount
of n-butyrate (Englyst et. al. (1987) in I. D. Morton: "Cereals in
a European Context", Chichester, UK, Ellis Horwood Ltd., pp.
221-223). This is considered important because of the general
acceptance that n-butyrate plays a major role in the prevention of
intestinal cancers (e.g. colorectal cancer) as recently summarised
by Smith and German (Food Technology, (1995 November) 87-90).
n-Butyrate appears to be a preferred substrate for normal
colonocytes and assists in the maintenance of colonic
integrity.
n-Butyrate inhibits growth of colon cancer cell lines. At the
molecular level, n-butyrate causes histone acetylation, favours
differentiation, induces apoptosis and regulates the expression of
various oncogenes. In vivo n-butyrate increases immunogenicity of
colon cancer cells.
Only indigestible polysaccharides which are associated with
production of high n-butyrate concentrations in the distal large
bowel (wheat bran, retrograded high amylose starch (type 3 RS))
were found to be protective against colorectal cancer in a rat
model system wherein rats were treated with 1,2-dimethylhydrazine
(McIntyre et al. (1993), Gut 34, 386-391; Young et al. (1996),
Gastroenterology 110(2): 508-514). Oat bran, guar gum, raw potato
starch (type 2 RS), cellulose and starch-free wheat bran have no
protective effect in this model of colorectal cancer (McIntyre et
al. (1993), Gut 34, 386-391, Young et al. (1996), Gastroenterology
110(2): 508-514).
From the above studies it appears that the amount of n-butyrate
produced in the colon is important. What is needed for a maximal
physiological benefit is not only a starch product with a high
amount of RS, but a well fermentable RS-fraction producing high
amounts of SCFA with an elevated n-butyrate level. Methods for the
preparation of resistant starch have for example been disclosed in
the following publications.
European patent application EP 688,872 discloses a method for
obtaining increased levels of resistant starch. It is demonstrated
that the highest amounts of RS are obtained when after enzymatic
digestion, retrogradation is performed for a prolonged period of
time and at a relatively low temperature. The maximum amount of RS
which could be obtained was 51.8% (example 3 therein).
International patent application WO 91/07106 discloses a method for
obtaining resistant starch wherein a retrogradation step is
followed by enzymatic hydrolysis. The retrogradation step is
performed at low temperature for amylose at 4.degree. C. and for
starch at 8.degree. C. as mentioned on page 13. Moreover the
process starts with undegraded starch which may be prior treated by
a debranching enzyme.
European patent application EP 564893 discloses a method for
obtaining resistant starch starting from a non-degraded high
amylose starch. The DSC melting peak temperature of this product is
mentioned to be in the range of 115-135.degree. C. and the amount
of resistant starch is below 51% and is correlated with the
percentage of amylose used in the starting product.
There exists a need for a starch-based product which is highly
fermentable and which gives rise to an increased amount of
n-butyrate in the colon. The present invention provides such a
starch-based product.
SUMMARY OF THE INVENTION
The present invention discloses a starch-based composition which is
characterised in that it contains a high amount of resistant
starch. The composition consists of partially degraded starch which
has undergone a retrogradation process and contains at least 55%
(w/w) pancreatine resistant starch. Preferably, the amount of
resistant starch is at least 60%. The resistant starch fraction is
characterised by a degree of polymerisation of predominantly
between 10 and 35 and a DSC peak temperature of below 115.degree.
C., preferably between 90 and 114.degree. C.
The partially degraded starch can be obtained by partial amylolytic
or acid hydrolysis of starch followed by enzymatic debranching. A
preferred partially degraded starch which is used as a starting
product is a maltodextrin with a dextrose equivalent (DE) below 10
obtained by partial alpha-amylase degradation and additionally
treated with isoamylase. The present invention also discloses a
method for obtaining the starch-based compositions. The method
comprises the following steps:
a) thinning of the starch,
b) enzymatic debranching of the thinned starch,
c) inactivation of the enzyme,
d) drying of the composition.
Step b) is preferably accompanied by retrogradation.
Preferably the high amount of resistant starch is obtained without
a separate retrogradation step at low temperature.
The present invention further discloses the use of the partially
degraded retrograded starches in the preparation of food or feed
compositions and food or feed compositions containing the
starch-based composition.
Finally, the invention discloses the use of partially degraded
retrograded starch composition to prevent or treat diseases of the
colorectal digestive tract.
DESCRIPTION OF THE FIGURES
FIG. 1 is an example of a DIONEX chromatogram of the resistant
starch fraction of the present invention obtained by exhaustive
pancreatine digestion of debranched potato maltodextrin (IRP)
(measured according to Carbohydr. Res. 215 (1991) 179-192).
FIG. 2 shows the change of pH in time during in vitro fermentation
of the RS fractions of Novelose.TM., Euresta and IRP.
FIG. 3 shows the formation of short-chain fatty acids during in
vitro fermentation of Novelose.TM., Euresta and IRP.
FIG. 4 shows the formation of n-butyrate during in vitro
fermentation of Novelose.TM., Euresta and IRP.
FIG. 5 shows DSC curves of milk drink residues obtained after
pancreatine digestion for standard milk and milk with added
IRP.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a starch-based composition which is
characterised in that it contains a high amount of resistant
starch. The composition consists of partially degraded starch which
has undergone a retrogradation process and contains at least 55%
(w/w) pancreatine resistant starch. Preferably, the amount of
resistant starch is at least 60%. The resistant starch fraction is
characterised by containing alpha-glucans with a degree of
polymerisation of predominantly between 10 and 35 and a DSC peak
temperature below 115.degree. C., preferably between 90-114.degree.
C. The partially degraded starch can be obtained by amylolytic or
acid degradation of starch followed by enzymatic debranching. A
preferred partially degraded starch is a maltodextrin obtained by
partial alpha-amylase degradation and treated with a debranching
enzyme.
The partially degraded starches for use as a starting material of
the present invention are obtainable from any suitable starch
source. Useful starches are obtained from potato, wheat, tapioca
and maize high amylose starches which are converted to
maltodextrins have also been used.
The present invention also discloses a method for obtaining the
starch-based compositions of this invention. The method comprises
the following steps:
a) thinning of the starch,
b) enzymatic debranching of the thinned starch,
c) inactivation of the enzyme,
d) drying of the composition.
The debranching is achieved by using a suitable enzyme such as
isoamylase or pullulanase, preferably by isoamylase
Step b) is preferably accompanied by retrogradation. Alternatively
the starch may be retrograded after enzyme inactivation.
A preferred process for obtaining the products of the present
invention contains the following steps:
a) maltodextrins (DE<10, preferably DE<5) are dissolved in
water,
b) the pH is adjusted and the solution is cooled to a optimum
temperature for the activity of a debranching enzyme,
c) the debranching enzyme is added and the mixture is
incubated,
d) the enzyme is inactivated,
e) the mixture is spray-dried
f) the resistant starch is collected and optionally grinded.
Preferably the maltodextrin is potato or tapioca maltodextrin.
The process of the present invention starts from a partially
degraded starch product. Contrary to known processes it was found
that no separate retrogradation step is required. Retrogradation
occurs at the same time as debranching. This results in a more
economical process as the retrogradation used to be performed
during a prolonged period (up to 48 hours) at a low temperature.
The process is therefore faster and cheaper.
Furthermore the product obtained by the invented process contains a
relatively high amount of resistant starch. It was found that this
product gives a higher production of n-butyrate both relatively
with respect to the other short chain fatty acids and in absolute
terms than other known products.
The present invention further discloses the use of the partially
degraded retrograded starches in the preparation of food or feed
compositions and food or feed compositions containing the
starch-based composition. The resistant starch product is added to
the food or feed composition in an amount of up to 20% (w/w),
preferably of up to 10%. Food preparations to which the
starch-based composition of the present invention is added include,
biscuits, toast, milk desserts up to 10% of the starch-based
composition of claim 1.
It is shown that heating during the preparation of the food product
does not significantly destroy the product. This means that
sufficient RS survives the treatment of food preparation including
UHT treatment and baking at 195.degree. C.
The invention also discloses the use of debranched/retrograded
maltodextrins in the prevention of diseases of the colorectal
digestive tract. On the basis of the finding that n-butyrate plays
a major role in the prevention of intestinal cancers (e.g.
colorectal cancer) as recently summarised by Smith and German (Food
Technology, 1995 (November) 87-90) the maltodextrins of the present
invention are expected, due to their production of a high amount of
n-butyrate, to assists in the maintenance of colonic integrity.
The present RS product having a specific chain length distribution
range of the retrograded structures is not only fermented more
easily but produces, in absolute and relative terms, significantly
higher amounts of n-butyrate than RS products derived from the
conventional high amylose starches.
In order to obtain the RS structures (after pancreatine treatment)
having more than 50% of the specified chain length of 10-35 AGU, a
suitable starting material is needed. We have found that a low DE
potato maltodextrin after enzymatic debranching and retrogradation
forms more than 30% RS, more preferably more than 40% RS. The RS
structures after pancreatine digestion consist for more than 50% of
linear chains of 10-35 alpha-glucans. Other debranched/retrograded
low DE maltodextrins (e.g. from tapioca, maize, wheat starch) can
be used for this purpose too as can maltodextrins obtained from
high amylose starches. Starches degraded by other methods (e.g.
acid thinned) followed by debranching/retrogradation are also
suited for this purpose.
Finally, the invention discloses the use of partially degraded
retrograded starch composition to prevent or treat diseases of the
colorectal digestive tract.
The invention is illustrated by the following examples.
Example 1 shows a method for obtaining the resistant starch of the
present invention. A commercial potato maltodextrin was dissolved
in water at an elevated temperature, after cooling and pH
adjustment the maltodextrin was debranched with isoamylase.
Following incubation the material was spray-dried. As shown in FIG.
1 the product had a chain length distribution wherein the majority
of the pancreatine resistant chains was between DP 10 and DP 35.
The resistant starch content was determined to be 56%.
This product is further indicated as IRP.
The experiment was repeated using tapioca maltodextrin and on a
larger scale. Starting with 4200 kg tapioca maltodextrin about 3500
kg spray-dried product was obtained which contained 66% resistant
starch had a DSC melting temperature of 112.degree. C. and
contained 65% material having a DP between 10 and 35.
Example 2 shows chemical and physical data for the resistant starch
of the present invention IRP in comparison with Euresta-RS and
Novelose.TM. (National Starch & Chemical Comp.). This example
demonstrates that IRP has a significantly higher content of
saccharide with DP 10-35 and much lower melting temperature of the
RS residues than the two other products.
Example 3 describes in vitro fermentation tests with three
different starch-based compositions;
IRP (obtained according to Example 1), Euresta and Novelose.TM.
(National Starch & Chemical Comp.). The pH of the fermentation
medium between the three RS products was found to be different
after 4 h of fermentation. The reduction of pH was more pronounced
for IRP than for Euresta and Novelose.TM. products. A slight
difference persisted after 8 h of fermentation. After 24 h of
fermentation pH values were identical (see FIG. 2). This indicates
that IRP is better fermentable than the other products. SCFA and
n-butyrate production were also followed. FIGS. 3 and 4 show the
amounts of SCFA and of n-butyrate formed during the fermentation of
the three samples. It appears that the IRP containing faeces gave
the highest amount of both SCFA and n-butyrate.
Example 4 describes the addition of resistant starch (IRP) to a
milk drink. It is demonstrated that after Ultra-High-Temperature
treatment RS can still be determined in the milk. It can be
concluded that RS can be applied in the normal food production
processes without the need of adaptation of this process.
The invention is further illustrated by the following non-limiting
examples.
EXAMPLE 1
Preparation of Debranched, Retrograded Low DE Maltodextrins
A) From Potato Maltodextrin
The reaction sequence for the production of the resistant starch
from potato maltodextrin according to the present invention is
given below.
Potato maltodextrin (MDx 01970 (DE 3) from Cerestar) was used as a
starting material. ##STR1##
It is evident that one does not have to start from a dried product
a wet product may directly be used in the same process. The
enzymatic debranching conditions corresponded to the conditions
given by the supplier for almost total debranching, about 59 units
of enzyme activity/g starch were used.
The product of the indicated process was characterised as
follows;
a resistant starch content of 56%,
a Mw of 11340,
a DSC melting peak temperature of the RS residue of 105.degree.
C.
the chain length distribution after pancreatine digestion of IRP is
shown in FIG. 1.
The major part of the resistant starch product consisted of
alpha-glucans with a DP between 10 and 35.
B) From Tapioca Maltodextrin
This example shows the large scale production of resistant starch
from a low DE tapioca (cassava) maltodextrin (DE 2.5) is described.
The debranching process was performed in a 20 m.sup.3 double wall
reactor. A freshly prepared maltodextrin was used after dilution to
25% d.s. The reaction scheme with more details is shown below:
##STR2##
The analysis of the 3.5 tons of spray-dried debranched maltodextrin
gave the following results:
a resistant starch content of 66%
a Mw of 7230
a DSC melting temperature of the RS residue of 112.degree. C.
a chain length distribution with 65% in the range between DP 10 and
35
EXAMPLE 2
Comparison of Composition and Properties of IRP with other RS
Products Based on Retrograded High Amylose Starches
The following table describes the RS content, the chain length
distribution and the DSC melting peak temperature of IRP,
Novelose.TM. and Euresta RS.
______________________________________ RS- DSC*- DP content**
T.sub.peak DP < 10*** 10-35*** DP > 35*** Sample (%)
(.degree. C.) (%) (%) (%) ______________________________________
IRP 56 105 7,0 58.7 34.3 Novelose 57 128 4,4 35,0 60,6 Euresta 36
141 5,1 26,1 68,8 ______________________________________ *DSC
measurement: 20-30 mg of starch was brought into a stainlesssteel
DSCpan and water was added to give a 20% (w/w) system. The closed
pan was heated in a SETARAM DSC 111 from 20-160.degree. C. at a
rate of 3.degree. C./min. The enthalpy change was continuously
recorded and the characteristic transition temperatures were
registered. **For RS determinations the following procedure was
used: A 5% (w/w) suspension of the retrograded starch product is
thoroughly homogenised in an acetate buffer solution. The acetate
buffer is made by dissolving 8.2 g anhydrous sodium acetate in 250
ml of a saturated aqueou solution of benzoic acid, adding 4 ml of
1M calcium chloride and making u to 800 ml with distilled water
before adjusting the pH to 5.2 with acetic acid, and finally making
up to 1000 ml # with distilled water. 25 ml of the suspension are
incubated with 1 ml pancreatic solution for 16 hours a 37.degree.
C. in a shaking water bath. The incubated suspension is next
stirred into 119 ml of 95% ethanol, filtered, the filter cake
washed twic with 80% ethanol and dried in an oven at 105.degree. C.
The RS content wa calculated as follows: #STR3## The pancreatic
solution is made by stirring 2 g pancreatine with 12 ml distilled
water for 10 min, centrifuging and using the supernatant as the
pancreatic solution. ***The saccharide distribution was analysed as
described in Carbohydr. Res. 215 (1991) 179-192 this method only
measures saccharides below DP 85 The content of saccharides DP >
was characterised by size exclusion chromatography and the relative
amounts of the different fractions were calculated after
normalisation.
EXAMPLE 3
In Vitro Fermentation of Partially Degraded Retrograded Starch
(IRP) in Comparison with Novelose.TM. and Euresta
Experimental
A. Starting Material
IRP, (Cerestar) was prepared according to Example 1. The product
was recovered by spray-drying. The product contained 56% RS and had
a DSC melting peak temperature of 105.degree. C.
Euresta-RS: retrograded starch was produced by cooling and storage
of extrusion-cooked amylomaize starch (Amylomaize VII, American
Maize Products Comp.). Amylomaize containing 50% water was
extrusion-cooked at 100.degree. C., followed by 4 days storage at
4.degree. C., then dried and milled. The product contained 36% RS
and had a DSC melting peak temperature of 141.degree. C.
Novelose.TM.: This starch is a modified amylomaize starch (National
Starch and Chemical Corp.), enriched in RS. It contains 57% RS and
had a DSC melting peak temperature of 128.degree. C.
B. Method Used for Predigestion of RS Products
The purification of the RS for in vitro fermentation has been
performed by extensive digestion of the starch with pancreatic
.alpha.-amylase (Sigma, A-3176).
42.5 g IRP, 72.7 g Euresta or 30.7 g of Novelose were suspended in
sterile phosphate buffer pH 6.9 (300 ml, 400 ml and 700 ml
respectively) and brought into a dialysis tube and .alpha.-amylase
(10 mg/g of sample) was added. The tubes were then plunged in 1 L
water at 37.degree. C. and kept overnight. The following day, the
same amount of .alpha.-amylase activity was added again and a
second digestion took place overnight. Samples were centrifuged (10
min, 3000 rpm) and washed several times. The sediment (RS) was
freeze-dried.
C. Method Used for the in Vitro Fermentations
The method has been extensively described elsewhere (Barry et al.,
Estimation of fermentability of dietary fibre in vitro: a European
interlaboratory study. Br. J. Nutr. (1995), 74, 303-322).
C1. General Schedule
All experiments were conducted in an in vitro batch system.
Fermentations were performed in vials using inoculum made from
fresh faeces collected from healthy young volunteers. The
volunteers usually ingested a normal diet, presented no digestive
disease and had not received antibiotics for at least three months.
Fermentation variables were measured in vials in which fermentation
was stopped at various times.
C2. Inoculum
Faeces from two non-methane producer volunteers were collected in
an insulated bottle previously warmed for about 5 min with hot tap
water (approximately 65.degree. C.). To eliminate O.sub.2, the
bottle was flushed for 5 min with CO.sub.2 at a flow of 100 ml/s
and faeces were then collected. When the insulated bottle was
received at the laboratory, CO.sub.2 was flushed inside. The weight
of faeces was then determined. The inoculum was produced in the
insulated bottle by adding five parts of a warmed (37.degree. C.)
nutritive buffer to one part of faeces (v/w). The nutritive medium
was made from carbonate-phosphate buffer solution containing (g/l):
NaHCO.sub.3 9.240, Na.sub.2 HPO.sub.4.12H.sub.2 O 7.125, NaCl
0.470, KCl 0.450, Na.sub.2 SO.sub.4 0.100, CaCl.sub.2 (anhydrous)
0.055, MgCl.sub.2 (anhydrous) 0.047, urea 0.400, with added trace
elements (10 ml of the following solution (mg/l) per liter of final
solution: FeSO.sub.4.7H.sub.2 O 3680, MnSO.sub.4.7H.sub.2 O1900,
ZnSO.sub.4.7H.sub.2 O 440, CoCl.sub.2.6H.sub.2 O 120,
CuSO.sub.4.5H.sub.2 O 98, Mo.sub.7 (NH.sub.4).sub.6 O.sub.24
4H.sub.2 O 17.4). Before use, and during preparation of the
inoculum, continuous bubbling of CO.sub.2 maintained anaerobiosis
and ensured a constant pH. The slurry was mixed using a Stomacher
(Laboratory Blended, Seward Medical, London) apparatus for 2 min
and then filtered through six layers of surgical gauze. The
inoculum was maintained in a water bath at 37.degree. C. and
continuously bubbled with CO.sub.2.
C3. Fermentation Experiments
Fermentation was conducted in duplicate using 50 ml polypropylene
vials (Falcon, Biolock). Except for blanks (B), 100 mg (dry-matter
basis) of well-homogenised experimental substrate was weighed into
each vial and 10 ml inoculum added. Air was displaced by flow of
O.sub.2 -free N.sub.2. After the cap was screwed on, the vial was
placed horizontally (time 0) in a shaking bath. Fermentation was
then performed at 37.degree. C. and the results studied at 0, 4, 8
and 24 h. Two blanks were used for each experimental time. At each
experimental time, fermentation in corresponding vials was stopped
by instantaneous freezing (dry ice).
C4. Sample Preparation
The pH was immediately measured and 10 ml distilled water added.
Sample was then centrifuged 10 min at 3000 g. Two samples of 1 ml
supernatant were taken for SCFA determinations. Samples were mixed
with 100 .mu.l HgCl.sub.2 -H.sub.3 PO.sub.4 (1%/5%) solution.
Samples for SCFA determination and pellets for starch
determinations were kept at -20.degree. C. until analysis.
SCFA were quantified by the gas chromatographic method as described
by Jouany J. P. (Dosage des acides gras volatils (AGV) et des
alcools dans les contenus digestifs, les jus d'ensilage, les
cultures bacteriennes et les contenus des fermenteurs anaerobies.
Sci. Alim., (1982), 2, 131-144).
Remaining starch was quantified by the method of Faisant et al.
(Resistant starch determination adapted to products containing high
resistant starch. Sci. Alim., (1995), 15, 83-89).
C5. Calculation of Short Chain Fatty Acid in Slurries
The production of P.sub.i of each SCFA was calculated as follows
for each experimental time:
where S.sub.i and S.sub.0 are SCFA concentration values in vials
containing substrates at time i and 0 respectively, and B.sub.i and
B.sub.0 are SCFA concentration values for blank at time i and 0
respectively.
For each experimental time, total SCFA production was calculated as
the sum of individual production of acetic, propionic and n-butyric
acid.
Results and Conclusion of in Vitro Fermentations
Kinetics of fermentation are determined by measuring pH and SCFA
production, in duplicate. For all parameters and each product the
same pattern of fermentation was observed upon comparing the
duplicate measurements.
A) Evolution of pH
The pH of the fermentation medium between the three RS products
differed after 4 h of fermentation. The reduction of pH was more
pronounced for IRP than for Euresta and Novelose products. A slight
difference persisted after 8 h of fermentation. After 24 h of
fermentation pH values were identical (see FIG. 2).
B) SCFA and N-butyrate Production
FIGS. 3 and 4 show the amount of SCFA and of n-butyrate formed
during the fermentation of the three samples. It appears that the
IRP containing faeces gave the highest amount of both SCFA and
n-butyrate. IRP gives rise to a faster production of n-butyrate
according to FIG. 4 more than 10 mMol/L was produced within 4
hours. FIG. 3 shows that also the amount of the other SCFA is
increased.
EXAMPLE 4
Preparation of UHT Milk Drinks with Resistant Starch
This example describes the use of the debranched retrograded
maltodextrin of example 1 in a UHT vanilla milk drink. The standard
recipe used for the preparation of milk drink is given below.
______________________________________ Standard recipe: whole milk
1000 ml ______________________________________ Satro mix* 12 g
Sucrose 20 g Dextrose 20 g ______________________________________
*Carrageenan, vanilla, colour
To this standard formula in one case 30 g/l and in the second case
60 g/l of IRP (see example 1) were added.
The ingredients were mixed and homogenised at 50 bar. The UHT
treatment was done with plate heating at 137.degree. C. for 5
seconds. The products were aseptically filled into 250 ml
bottles.
After cooling to ambient temperature the products were
characterised:
______________________________________ Product RS-content (%) Taste
and mouthfeel ______________________________________ Standard 0,0
very liquid, no off-taste, no sandiness + 30 g 1,0 somewhat more
mouthfeel, more creamy, IRP/l no off-taste, no sandiness + 60 g
1,95 most mouthfeel, no sandiness IRP/l
______________________________________
The results show that the major RS part survives even
UHT-processing and is detectable in the final product using the
method as mentioned in example 3. This is further confirmed by the
DSC measurement (for method see example 2) of the residues obtained
after pancreatine digestion (see FIG. 5). The sample prepared with
60 g/l IRP shows a strong endothermic transition with a peak
temperature around 96.degree. C. whilst the standard product does
not show any significant transition in this temperature range. The
use of IRP does not only increase the RS content but improves the
organoleptic properties to a significant extent. Due to the small
particle size there is no sandiness and the use of IRP causes the
impression of a higher fat content. IRP can therefore be used with
advantage in low (no) fat products in order to improve the
sensorial properties.
* * * * *